Abstract Several approaches are available to increase the efficiency of CRISPR/Cas9-based genome editing, including the co-inactivation of a gene that mediates the cytotoxic activity of a compound which can be used to enrich the population in edited cells. Here we show in multiple cell lines how inactivating HSD17B11, a non-essential Short-chain Dehydrogenase/Reductase, confers a strong resistance (29- to 130-fold resistance) in both human and mouse cells to a Phenyl diAlkynylCarbinol compound (PAC) without impacting cell viability and proliferation. We show how co-inactivating HSD71B11 along with selection with PAC is usable to quickly identify efficient guide(s) against a gene of interest and to readily isolate fully inactivated clones. Altogether, this work provides an experimental framework for the facile generation of knockouts using PAC for selecting successfully inactivated cells.

Malaria caused by Plasmodium falciparum remains a major health threat, killing over 600,000 people annually. The spread of resistance to all major antimalarials, including artemisinins, highlights the urgent need for new drugs with distinct mechanisms of action. Here we show that the G-quadruplex ligand RHPS4, an acridine derivative, displays strong antiplasmodial activity against both drug-sensitive and -resistant P. falciparum strains and clinical isolates. RHPS4 primarily targets the trophozoite stage and induces major mitochondrial alterations, including reduction of mitochondrial DNA (mtDNA) and transcriptional dysfunctions. Bioinformatic analyses identified at least eight putative G4-forming sequences within the parasite’s mtDNA. Biophysical studies confirmed G4 folding of at least one sequence and its interaction with RHPS4. These findings indicate that RHPS4 disrupts P. falciparum mitochondrial metabolism through G4 stabilization, leading to parasite death, and establish mtDNA G4 structures as novel therapeutic targets for antimalarial development. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Werner (WRN) is the only human RecQ helicase family member with DNA exonuclease activity. WRN promotes genome stability through its functions in DNA replication, repair and telomere maintenance, the deficiency of which presents clinically as Werner syndrome, causing premature aging and cancer predisposition. The main DNA double strand-break sensor Ku70/80 heterodimer (Ku) is a known partner of WRN, which stimulates its nuclease activity. However, the molecular basis of Ku-WRN interplay is currently unknown. Here, we present a high resolution cryo-EM structure of human Ku bound to DNA in complex with the N-terminal WRN exonuclease domain. This structure reveals multiple interaction sites between WRN and the Ku:DNA complex. The catalytic domain of WRN-exo engages with the DNA ends, stabilized by the vWA-like Ku80 domain interacting with the N-terminal APLF-like Ku binding motif (A-KBM) of WRN. Most surprisingly, we visualize the SAP domain of Ku70 stabilized within this complex, and we identify specific contacts mediating this interaction. These interactions are validated by assessing the impact of point mutations on either side of the Ku-WRN interfaces on exonuclease activity with purified recombinant proteins, and on live protein recruitment at biphoton laser-damaged nuclear sites. Finally, we show that disruption of WRN-Ku70 interaction results in aberrant resection of stalled replication forks. Together, we define the architecture of the Ku-WRN exonuclease domain interface and its impact on WRN exonuclease activity, recruitment and replication fork processing.

G-quadruplexes (G4s) are four-stranded nucleic acid structures that have gathered a significant attention due to their involvement in key biological processes, including gene regulation, genome stability, and telomeres maintenance. Some G4 antibodies have been developed to selectively recognize these structures over duplex DNA; however, most, even the widely studied BG4 and 1H6, bind G4s in a general manner and lack discrimination between distinct topologies, particularly between parallel and antiparallel conformations. In this study, we report on the development and characterization of a novel antibody selected via phage display method using a constrained antiparallel G4 structure mimicking one of the conformation adopted in vitro by the human telomeric sequence. Our findings demonstrate that this new antibody selectively recognizes the antiparallel topology of the telomeric G4 sequence, a property further validated in cellular models. ### Competing Interest Statement The authors have declared no competing interest. French National Centre for Scientific Research, https://ror.org/02feahw73, ANR-16-CE11-0006-01, ANR-21-CE44-0005-02, ANR-17-EURE-0003, ANR-24-INBS-0005 FBI BIOGEN

Treacle ribosome biogenesis factor 1 ( TCOF1 ) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with K d values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter ( nolc1 ). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD.

i-Motifs (iMs) are non-canonical, four-stranded secondary structures formed by stacking of hemi-protonated CH+·C base pairs in cytosine-rich DNA sequences, predominantly at pH < 7. The presence of iM structures in cells was a matter of debate until the recent development of iM-specific antibody, iMab, which was instrumental for several studies that suggested the existence of iMs in live cells and their putative biological roles. We assessed the interaction of iMab with cytosine-rich oligonucleotides by biolayer interferometry (BLI), pull-down assay and bulk-FRET experiments. Our results suggest that binding of iMab to DNA oligonucleotides is governed by the presence of runs of at least two consecutive cytosines and is generally increased in acidic conditions, irrespectively of the capacity of the sequence to adopt, or not, an iM structure. Moreover, the results of the bulk-FRET assay indicate that interaction with iMab results in unfolding of iM structures even in acidic conditions, similarly to what has been observed with hnRNP K, well-studied single-stranded DNA binding protein. Taken together, our results strongly suggest that iMab actually binds to blocks of 2–3 cytosines in single-stranded DNA, and call for more careful interpretation of results obtained with this antibody.

Figure S3 shows the monitoring of the levels of TOP2α cleavage complexes in cells by the ICE bioassay.

G-quadruplexes (G4) are non-canonical DNA structures found in the genome of most species including human. Small molecules stabilizing these structures, called G4 ligands, have been identified and, for some of them, shown to induce cytotoxic DNA double-strand breaks. Through the use of an unbiased genetic approach, we identify here topoisomerase 2α (TOP2A) as a major effector of cytotoxicity induced by two clastogenic G4 ligands, pyridostatin and CX-5461, the latter molecule currently undergoing phase I/II clinical trials in oncology. We show that both TOP2 activity and transcription account for DNA break production following G4 ligand treatments. In contrast, clastogenic activity of these G4 ligands is countered by topoisomerase 1 (TOP1), which limits co-transcriptional G4 formation, and by factors promoting transcriptional elongation. Altogether our results support that clastogenic G4 ligands act as DNA structure-driven TOP2 poisons at transcribed regions bearing G4 structures.

G-quadruplexes (G4) are non-canonical secondary structures consisting in stacked tetrads of hydrogen-bonded guanines bases. An essential feature of G4 is their intrinsic polymorphic nature, which is characterized by the equilibrium between several conformations (also called topologies) and the presence of different types of loops with variable lengths. In cells, G4 functions rely on protein or enzymatic factors that recognize and promote or resolve these structures. In order to characterize new G4-dependent mechanisms, extensive researches aimed at identifying new G4 binding proteins. Using G-rich single-stranded oligonucleotides that adopt non-controlled G4 conformations, a large number of G4-binding proteins have been identified in vitro, but their specificity towards G4 topology remained unknown. Constrained G4 structures are biomolecular objects based on the use of a rigid cyclic peptide scaffold as a template for directing the intramolecular assembly of the anchored oligonucleotides into a single and stabilized G4 topology. Here, using various constrained RNA or DNA G4 as baits in human cell extracts, we establish the topology preference of several well-known G4-interacting factors. Moreover, we identify new G4-interacting proteins such as the NELF complex involved in the RNA-Pol II pausing mechanism, and we show that it impacts the clastogenic effect of the G4-ligand pyridostatin.

G-quadruplexes (G4), non-canonical DNA structures, are involved in several essential processes. Stabilization of G4 structures by small compounds (G4 ligands) affects almost all DNA transactions, including telomere maintenance and genomic stability. Here, thanks to a powerful and unbiased genetic approach, we identify topoisomerase 2-alpha (TOP2A) as the main effector of cell cytotoxicity induced by CX5461, a G4 ligand currently undergoing phase I/II clinical trials. This approach also allowed to identify new point mutations affecting TOP2A activity without compromising cell viability. Moreover, based on cross-resistance studies and siRNA-based protein depletion we report that TOP2A plays a major role in cell cytotoxicity induced by two unrelated clastogenic G4 ligands, CX5461 and pyridostatin (PDS). We also report that cytotoxic effects induced by both compounds are associated with topoisomerase 2-mediated DNA breaks production. Finally, we show that TOP2-mediated DNA breaks production is strongly associated with RNA Pol II-dependent transcription and is countered by topoisomerase 1 (TOP1). Altogether our results indicate that clastogenic G4 ligands act as DNA structure-driven TOP2-poisons at transcribed regions bearing G-quadruplex structures.

Guanine-rich DNA strands can fold into non-canonical four-stranded secondary structures named G-quadruplexes (G4). Experimental evidences suggest that G4-DNA surrounding transcription start sites act as cis-regulatory elements by either stimulating or inhibiting gene transcription. Therefore, proteins able to target and regulate specific G4 formation/unfolding are crucial for G4-mediated transcriptional control. Here we present data revealing that CNBP acts in vitro as a G4-unfolding protein over a tetramolecular G4 formed by the TG(4)T oligonucleotide, as well as over the G4 folded in the promoters of several oncogenes. CNBP depletion in cellulo led to a reduction in the transcription of endogenous KRAS, suggesting a regulatory role of CNBP in relieving the transcriptional abrogation due to G4 formation. CNBP activity was also assayed over the evolutionary conserved G4 enhancing the transcription of NOGGIN ( NOG) developmental gene. CNBP unfolded in vitro NOG G4 and experiments performed in cellulo and in vivo in developing zebrafish showed a repressive role of CNBP on the transcription of this gene by G4 unwinding. Our results shed light on the mechanisms underlying CNBP way of action, as well as reinforce the notion about the existence and function of G4s in whole living organisms.

Porphyrins represent a valuable class of ligands for G-quadruplex nucleic acids. Herein, we evaluate the binding of cationic porphyrins metallated with gold(iii) to G-quadruplex DNA and we compare it with other porphyrin derivatives. The G-quadruplex stabilization capacity and the selectivity of the various porphyrins were evaluated by biophysical and biochemical assays. The porphyrins were also tested as inhibitors of telomerase. It clearly appeared that the insertion of gold(iii) ion in the center of the porphyrin increases the binding affinity of the porphyrin for the G-quadruplex target. Together with modelling studies, it is possible to propose that the insertion of the square planar gold(iii) ion adds an extra positive charge on the complex and decreases the electron density in the porphyrin aromatic macrocycle, both properties being in favour of stronger electrostatic and π-staking interactions.

G-quadruplexes (G4) are RNA and DNA secondary structures formed by the stacking of guanine quartets in guanine rich sequences. Quadruplex-prone motifs may be found in key genomic regions such as telomeres, ribosomal DNA, transcriptional activators and regulators or oncogene promoters. A number of proteins involved in various biological processes are able to interact with G4s. Among them, proteins dedicated to nucleic acids unwinding such as WRN, BLM, FANCJ or PIF1, can unfold G4 structures. Mutations of these helicases are linked to genome instability and to increases in cancer risks. Here, we present a high-throughput fluorescence-based reliable, inexpensive and fast assay to study G4/RHAU interaction. RHAU is an RNA helicase known as the major source of G4 resolution in HeLa cells. Our assay allows to monitor the unfolding properties of RHAU towards DNA and RNA quadruplexes in parallel and to screen for the optimal conditions for its activity. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Abstract Poisons of topoisomerase II (TOP2) kill cancer cells by preventing religation of intermediate DNA breaks during the enzymatic process and thus by accumulating enzyme–drug–DNA complexes called TOP2 cleavage-complex (TOP2cc). F14512 is a highly cytotoxic polyamine-vectorized TOP2 inhibitor derived from etoposide and currently in clinical trials. It was shown in vitro that F14512 has acquired DNA-binding properties and that the stability of TOP2cc was strongly increased. Paradoxically, at equitoxic concentrations in cells, F14512 induced less DNA breaks than etoposide. Here, we directly compared etoposide and F14512 for their rates of TOP2cc production and resolution in human cells. We report that targeting of TOP2α and not TOP2β impacts cell killing by F14512, contrary to etoposide that kills cells through targeting both isoforms. Then, we show that despite being more cytotoxic, F14512 is less efficient than etoposide at producing TOP2α cleavage-complex (TOP2αcc) in cells. Finally, we report that compared with TOP2αcc mediated by etoposide, those generated by F14512 persist longer in the genome, are not dependent on TDP2 for cleaning break ends from TOP2α, are channeled to a larger extent to resection-based repair processes relying on CtIP and BRCA1 and promote RAD51 recruitment to damaged chromatin. In addition to the addressing of F14512 to the polyamine transport system, the properties uncovered here would be particularly valuable for a therapeutic usage of this new anticancer compound. More generally, the concept of increasing drug cytotoxicity by switching the repair mode of the induced DNA lesions via addition of a DNA-binding moiety deserves further developments. Mol Cancer Ther; 16(10); 2166–77. ©2017 AACR.

Four nickel(II)-salophen complexes containing alkyl-imidazolium chains connected at the ortho or meta positions were prepared: N,N'-bis(2-hydroxy-4-methyl-3H-imidazol-1-iumbenzylideneamino) phenylenediamine (1), N, N'-bis(2-hydroxy-3-methyl-3H-imidazol-1-iumbenzylideneamino) phenylenediamine (2), N, N'-bis(2-hydroxy-3-methyl-3H-imidazol-1-iumbenzylideneamino) methyl-3H-imidazol-1-iumphenylenediamine (3), and N,N'bis(2-hydroxy-4-methyl-3H-imidazol-1-iumbenzylideneamino)methyl-3H-imidazol-1-iumphenylenediamine (4). They protect G-quadruplex DNA (G4-DNA) against thermal denaturation and show K-A values in the range of 7.4 x 10(5) to 4 x 10(7) m(-1) for G(4)-DNA models. Complex 4 exhibits an IC50 value of 70 nm for telomerase inhibition.

With the aim of finding selective and biologically active G-quadruplex ligands, modified porphyrin with bulky cationic substituents, meso-5,10,15,20-tetrakis(4-guanidinophenyl)porphyrin tetrahydrochloride, referred to as guanidinium phenyl porphyrin, was prepared. The corresponding nickel(II) and cobalt(III) metallated porphyrins were also synthesized. Interaction with quadruplexes was examined by means of fluorescence resonance energy transfer melting and surface plasmon resonance-based assays: the three compounds proved to bind to G-quadruplex DNA in a similar and highly selective way. Guanidinium phenyl porphyrin and its nickel(II) metallated derivative exhibit moderate cytotoxicity toward cells in culture. Strikingly, the nickel porphyrin derivative was able to displace hPOT1 shelterin protein from telomeres in human cells.Nickel(II) guanidinium phenyl porphyrin, a cationic bulky porphyrin is a powerful specific G-quadruplex DNA ligand. It enters the cells and induces shelterin modification.

A series of nine Ni(II) salophen complexes involving one, two, or three alkyl-imidazolium side-chains was prepared. The lengths of the side-chains were varied from one to three carbons. The crystal structure of one complex revealed a square planar geometry of the nickel ion. Fluorescence resonance energy transfer melting of G-quadruplex structures in the presence of salophen complex were performed. The G-quadruplex DNA structures were stabilized in the presence of the complexes, but a duplex DNA was not. The binding constants of the complexes for parallel and antiparallel G-quadruplex DNA, as well as hairpin DNA, were measured by surface plasmon resonance. The compounds were selective for G-quadruplex DNA, as reflected by equilibrium dissociation constant KD values in the region 0.1-1 μM for G-quadruplexes and greater than 2 μM for duplex DNA. Complexes with more and shorter side-chains had the highest binding constants. The structural basis for the interaction of the complexes with the human telomeric G-quadruplex DNA was investigated by computational studies: the aromatic core of the complex stacked over the last tetrad of the G-quadruplex with peripherical cationic side chains inserted into opposite grooves. Biochemical studies (telomeric repeat amplification protocol assays) indicated that the complexes significantly inhibited telomerase activity with IC50 values as low as 700 nM; the complexes did not significantly inhibit polymerase activity.

To cope with DNA double strand break (DSB) genotoxicity, cells have evolved two main repair pathways: homologous recombination which uses homologous DNA sequences as repair templates, and non-homologous Ku-dependent end-joining involving direct sealing of DSB ends by DNA ligase IV (Lig4). During the last two decades a third player most commonly named alternative end-joining (A-EJ) has emerged, which is defined as any Ku- or Lig4-independent end-joining process. A-EJ increasingly appears as a highly error-prone bricolage on DSBs and despite expanding exploration, it still escapes full characterization. In the present review, we discuss the mechanism and regulation of A-EJ as well as its biological relevance under physiological and pathological situations, with a particular emphasis on chromosomal instability and cancer. Whether or not it is a genuine DSB repair pathway, A-EJ is emerging as an important cellular process and understanding A-EJ will certainly be a major challenge for the coming years.